Journal of General Virology (2006), 87, 1685–1690
Short
Communication
DOI 10.1099/vir.0.81692-0
Feline infectious peritonitis virus-infected
monocytes internalize viral membrane-bound
proteins upon antibody addition
Hannah L. Dewerchin, Els Cornelissen and Hans J. Nauwynck
Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133,
9820 Merelbeke, Belgium
Correspondence
Hans J. Nauwynck
[email protected]
Received 17 November 2005
Accepted 7 February 2006
Feline infectious peritonitis virus (FIPV) may cause a highly lethal infection in cats, in spite of a
usually strong humoral immune response. Antibodies seem unable to identify infected cells and
mediate antibody-dependent cell lysis. In this study, the effect of antibodies on Feline coronavirus
(FCoV)-infected monocytes was investigated. Upon addition of FCoV-specific antibodies,
surface-expressed viral proteins were internalized through a highly efficient process, resulting in
cells without visually detectable viral proteins on their plasma membrane. The internalization was
also induced by mAbs against the Spike and Membrane proteins, suggesting that both proteins
play a role in the process. The internalization did not occur spontaneously, as it was not observed
in cells incubated with medium or non-specific antibodies. Further, the internalization could not
be reproduced in feline cell lines, indicating its cell-type specificity. This study sheds new light on
the immune-evasive nature of FIPV infections.
Feline infectious peritonitis virus (FIPV) and feline enteric
coronavirus (FECV) are two coronaviruses described in cats.
These feline coronaviruses are spread worldwide and infect
all members of the family Felidae. Very little is known about
the interactions of FIPV with the host immune system. Cats
with clinical feline infectious peritonitis (FIP) often have
very high titres of FIPV-specific antibodies; however, these
antibodies are not able to block infection. This suggests
that, for unknown reasons, antibodies and antibody-driven
immune effectors are not able to clear the body of virus
and/or virus-infected cells efficiently. There are indications
that the immune system even plays an adverse role in the
development of FIP. It has been reported in experimental
infections that cats that have obtained FIPV-specific antibodies actively or passively develop FIP faster and more
severely than naive cats (Pedersen & Boyle, 1980). This
accelerated FIP has been the reason for the failure of most
vaccination attempts (Woods & Pedersen, 1979; Pedersen &
Black, 1983; Barlough et al., 1984, 1985; Vennema et al.,
1990; McArdle et al., 1992). A mechanism was proposed that
could explain this accelerated development of FIP in the
presence of antibodies: antibody-dependent enhancement
of infectivity (ADEI) (Hohdatsu et al., 1991; Corapi et al.,
1992; Olsen et al., 1992). ADEI suggests that antibodies
might help the spread of the virus in an infected cat by
facilitating the virus uptake through the formation of virus–
antibody complexes that are taken up by uninfected monocytes/macrophages via the Fc receptor. ADEI may explain
why a larger number of cells can be infected in the presence
of antibodies, but it cannot explain why these infected cells
0008-1692 G 2006 SGM
are not eliminated by the immune system. It is believed that
the only effective defence against FIP is cell-mediated
immunity (Pedersen, 1987).
The role of antibodies in the pathogenesis of naturally
occurring FIP and, more specifically, how antibodies interact with infected cells is unknown. In the present study, we
investigated the effect of FCoV-specific antibodies on FCoVinfected monocytes to clarify why antibodies seem to be
unable to identify infected cells and/or mark them for
antibody-dependent cell lysis. Feline coronavirus (FCoV)-,
Feline leukemia virus (FeLV)- and Feline immunodeficiency
virus (FIV)-negative cats were used as blood donors. Monocytes were isolated as described previously (Dewerchin et al.,
2005) and seeded on glass coverslips, which allowed mounting on microscope slides using glycerin/DABCO (Janssen
Chimica). The adherent cells consisted of 86±7 % monocytes (assessed with monocyte marker DH59B; Veterinary
Medical Research and Development). At 36 h post-seeding,
monocytes were inoculated at an m.o.i. of 5 with third
passages of FIPV 79-1146 and FECV 79-1683 on Crandell
feline kidney (CrFK) cells (McKeirnan et al., 1981). FIPV
79-1146 was obtained from the ATCC and FECV 79-1683
was kindly provided by Dr Egberink (Utrecht University,
The Netherlands). Twelve hours after inoculation, monocytes were incubated with feline biotinylated anti-FCoV
polyclonal antibodies (kindly provided by Dr Egberink). At
different times post-antibody addition, cells were fixed with
1 % formaldehyde, permeabilized with 0?1 % Triton X-100
(Sigma-Aldrich) and incubated with streptavidin–fluorescein
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H. L. Dewerchin, E. Cornelissen and H. J. Nauwynck
isothiocyanate (FITC) (Molecular Probes). In order to find
the infected cells in the population easily, the cells were
incubated with a mixture of mAbs 7-4-1, F19-1 and E22-2,
recognizing respectively the Spike (S), Membrane (M)
and Nucleocapsid (N) proteins (kindly provided by Dr
Hohdatsu, Kitasato University, Japan), and visualized with
goat anti-mouse–Texas red (Molecular Probes) (not shown).
For the controls, cells were incubated with non-specific
polyclonal antibodies that were obtained from specificpathogen-free cats vaccinated with Nobivac Tricat (Intervet).
After fixation of the cells, surface expression of viral proteins
was visualized by a subsequent incubation with a mixture of
anti-S and anti-M mAbs and goat anti-mouse–Texas red.
Next, the cells were permeabilized and incubated with goat
anti-cat–FITC (Sigma-Aldrich) to visualize possible internalization caused by the non-specific polyclonal antibodies (not shown). In Fig. 1(a), confocal images illustrate
that, after FCoV-specific antibody addition, the surfaceexpressed viral proteins moved from the plasma membrane
into the cytoplasm. In contrast, after addition of nonspecific antibodies, the surface-expressed viral proteins
remained in the plasma membrane. Fig. 1(c) shows that
internalization of the viral glycoproteins was initiated very
shortly after antibody addition and was completed rapidly.
Fig. 1. Internalization assays with polyclonal antibodies. (a) Images show the localization of surface-expressed viral proteins in
FCoV-infected monocytes at different times after addition of a-FCoV or non-specific polyclonal antibodies. (b) Spontaneousinternalization assay. The distribution of surface-expressed proteins was visualized at 0 or 30 min incubation with RPMI
medium, a-FCoV or non-specific polyclonal antibodies. All images are a single section through the cell. Bar, 5 mm.
(c) Internalization of surface-expressed viral proteins in FIPV- or FECV-infected monocytes after addition of polyclonal
antibodies. $, FIPV; #, FECV; ,, non-specific polyclonal antibody. Data represent means±SD of triplicate assays.
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Journal of General Virology 87
Internalization of FIPV surface-expressed proteins
In the graph, the internalization was represented as the
percentage of cells that were internalizing viral proteins and
not as number of internalized antibody–antigen complexes
per cell, because the amount of viral proteins that is
expressed in the plasma membrane varies strongly between
cells (Dewerchin et al., 2005). The curves indicate that
89±9 and 84±4 % of respectively FIPV- and FECVinfected monocytes showed internalization of the plasma
membrane-bound viral proteins after 3 min incubation.
At 30 min, almost 100 % of the infected monocytes internalized their membrane-bound proteins (98±3 and
97±4 % for FIPV and FECV infection, respectively). Considering that an immune-evasive nature is attributed only
to FIPV, it was remarkable to find that FIPV and FECV show
almost-identical internalization kinetics. These identical
kinetics imply that the difference between FIPV and FECV
pathogenesis cannot be explained by the ability to internalize viral proteins. Fig. 1(c) also shows that internalization
was not observed after addition of non-specific antibodies.
This indicates that specific Fab–antigen interactions are
needed and that the internalization is not mediated by the
Fc-binding capacity that has been described for the S protein of several coronaviruses (Oleszak et al., 1993). To
confirm that the membrane-bound structures were single
proteins and not virus particles, infected cells were fixed
and membrane expression was visualized. Then, the cells
(and virus membrane) were permeabilized and nucleocapsid proteins were stained. No co-localization was found
between the nucleocapsid and membrane-bound proteins,
which shows that the structures were single proteins (data
not shown).
Next, a spontaneous-internalization assay was performed.
In this assay, it was tested whether internalization could
occur spontaneously and whether it could be induced by
non-specific cat antibodies. For this assay, all surfaceexpressed proteins were labelled with biotin. Monocytes, at
12 h after inoculation, were placed on ice, washed twice
with ice-cold PBS solution and incubated with 2 mM of
the biotinylation reagent EZ-Link sulfo-NHS-LC-Biotin
(Pierce). After 30 min, the biotin was removed and replaced
by cold medium supplemented with 10 mM glycine for
10 min. Then, the monocytes were washed twice with cold,
supplemented medium and twice with cold medium without fetal bovine serum or heparin. Cells were shifted to 37 uC
and incubated with anti-FIPV polyclonal antibodies, nonspecific antibodies or RPMI medium for 30 min. Control
cells were fixed before the temperature shift. To visualize
internalized, biotinylated proteins, cells were fixed, permeabilized and incubated with streptavidin–Texas red (Molecular
Probes). Afterwards, cells were incubated with polyclonal
feline anti-FIPV–FITC (VMRD) to enable identification of
infected cells (not shown). Fig. 1(b) shows that a temperature shift by itself did not lead to internalization; nor did
incubation with non-specific antibodies. Only monocytes
that were incubated with anti-FCoV antibodies showed
internalized proteins. These results indicate that spontaneous internalization did not occur.
http://vir.sgmjournals.org
To further specify which membrane-bound viral proteins
are of importance for the internalization process, the redistribution of proteins induced by mAbs directed against
the S or M protein was studied. At 12 h after inoculation,
monocytes were incubated with anti-S (7-4-1, subisotype
IgG2b), anti-M (F19-1, subisotype IgG1) or a combination
of both antibodies. At different times post-antibody addition, cells were fixed, permeabilized and incubated with goat
anti-mouse–Texas red to visualize the distribution of the
antigen–antibody complexes. Next, the cells were incubated with FITC-labelled anti-FIPV antibodies to allow easy
recognition of infected cells (not shown). The confocal
images in Fig. 2(a) illustrate that both anti-S and anti-M
antibodies were able to induce internalization. Fig. 2(b)
shows that 82±9 and 66±4 % of the infected cells showed
internalization at 10 min after addition of anti-S or anti-M
antibodies, respectively. These percentages further increased
and, after 1 h incubation with anti-S or anti-M antibodies,
respectively 85±4 and 81±4 % of the cells showed internalization. The results demonstrate that internalization
induced by mAbs occurred less efficiently than internalization induced by polyclonal anti-FIPV antibodies. Incubation
with both anti-S and anti-M antibodies led to internalization in 100 % of infected monocytes; thus, the same efficiency was reached as with polyclonal antibodies. In cells
where all antigen–antibody complexes were internalized
with one monoclonal antibody against S or M protein, no
residual expression could be found in the plasma membrane
by using a polyclonal antibody (data not shown). In cells
where not all complexes were internalized with one mAb
against the S or M protein, the complexes that were in the
plasma membrane could also be stained for the other protein (data not shown). These results indicate that S and M
proteins reside in the plasma membrane as complexes.
Interactions between the S and M proteins have already been
described in mouse hepatitis virus infection, during which
the M and S proteins form heteromultimeric complexes
(Opstelten et al., 1995). Taken together, these findings
suggest that S and M proteins operate together to mediate
the internalization process. Internalization could not be
induced by using anti-N antibodies (data not shown). As the
mAbs against S and M protein are of mouse origin, nonspecific mouse monoclonals of the same isotype were tested
as a control in order to exclude isotype-specific interactions.
Inoculated monocytes were incubated for different time
periods with a mixture of non-specific, isotype-matched
mAbs: 41D3 (isotype IgG1), recognizing porcine sialoadhesin, and Mil2 (isotype IgG2b), recognizing porcine CD14
(Duan et al., 1998; Thacker et al., 2001; Vanderheijden et al.,
2003). After fixation of the cells, surface expression of viral
proteins was visualized by incubation with biotinylated antiFCoV polyclonal antibodies and then streptavidin–Texas
red (Molecular Probes). Next, the cells were permeabilized
and incubated with goat anti-mouse–FITC (Sigma-Aldrich)
to visualize possible internalization caused by the nonspecific mAbs. No internalized, non-specific antibodies
were found (not shown). Fig. 2(a) shows that addition of
non-specific antibodies did not lead to internalization,
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H. L. Dewerchin, E. Cornelissen and H. J. Nauwynck
Fig. 2. Internalization assays with mAbs. (a) Images show the localization of surface-expressed viral proteins after addition of
monoclonal anti-S (IgG2b) or anti-M (IgG1) antibodies, a combination of both or non-specific, isotype-matched mAbs 41D3
(IgG1) and Mil2 (IgG2b). The images show a section through the cells. Bar, 5 mm. (b) Internalization of surface-expressed viral
proteins in FIPV-infected monocytes after addition of monoclonal antibodies. $, Anti-S + anti-M mAb; ,, anti-S mAb; .,
anti-M mAb; #, non-specific mAbs. Data represent means±SD of triplicate assays.
confirming that the internalization process requires FCoVspecific antibodies and is not mediated by isotype-specific
interactions.
Commonly used feline cell lines, CrFK cells and the
macrophage-like ‘Felis catus whole fetus’ (fcwf) cells, were
tested for their ability to internalize surface-expressed viral
proteins. For this, cells at 12 h post-inoculation were incubated with biotinylated anti-FCoV polyclonal antibodies. At
30 min post-antibody addition, cells were fixed, permeabilized and incubated with streptavidin–Texas red. Then,
cytoplasmic expression of antigens was visualized with antiFIPV–FITC (not shown). Fig. 3 shows that the cell lines
expressed viral proteins on their cell surface and that, after
addition of antibodies, the antigens were somewhat more
clustered than in non-treated cells. However, internalization
was not observed. These findings suggest that the internalization process requires cellular machinery that is not, or
not completely, present in cell lines. It also means that a
more thorough study of this internalization pathway using
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Fig. 3. The distribution of surface-expressed viral proteins upon
antibody addition in CrFK and fcwf cells. The images show a
section through the middle of the cells. Bar, 5 mm.
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Journal of General Virology 87
Internalization of FIPV surface-expressed proteins
dominant-negative mutants will be hampered by the lack of
adequate cell lines.
In this study, a mechanism is presented that might aid
in explaining why the humoral immune system is not
effective against an FIPV infection: internalization of viral
plasma membrane-bound proteins induced by antibodies.
This immune-evasion mechanism was described for the
first time by Favoreel et al. (1999). They found that viral
plasma membrane-bound proteins in Pseudorabies virus
(PrV)-infected pig monocytes were internalized upon
antibody addition. This internalization process is clathrinmediated and dependent on a YXXY motif (Y stands for
tyrosine, X for any amino acid and Y for a bulky hydrophobic amino acid) in the cytoplasmic tail of the gB protein
(Van de Walle et al., 2001; Favoreel et al., 2002). The viral
plasma membrane-bound proteins in FIPV-infected cells
(S and M) contain putative internalization motifs in their
cytoplasmic tails. The S protein contains a dileucine motif
and a YXXY motif and the M protein contains two of each.
The presence of these putative internalization motifs is
another indication that both viral proteins are of importance in antibody-mediated internalization. The role of
these motifs will be investigated in the future.
In previous work, we reported that only half of FIPVinfected monocytes express viral proteins on their plasma
membrane (Dewerchin et al., 2005). Here, we report that
cells that do express viral proteins internalize these proteins
upon antibody addition. With these findings, the following
hypothetical model may aid in explaining FIP pathogenesis.
In an FIPV-infected cat, a proportion of the FIPV-infected
monocytes may remain immune-masked because no viral
antigens are expressed at the plasma membrane and a proportion of the cells may express viral proteins. When antibodies bind to these membrane-bound proteins to mark the
infected cells for cell lysis, internalization may be triggered.
The plasma membrane is cleared of viral proteins and the
infected cell remains invisible to the humoral immune system.
In this way, the cell may be able to continue the production
of progeny virus without being eliminated or it may enter a
quiescent-infection state, as is seen in PrV-infected monocytes that were cultured in the presence of specific antibodies
(Favoreel et al., 2003). This quiescent infection state would be
an excellent cover for a carrier cell and might explain the
sometimes long incubation period of an FIPV infection. For
PrV-infected pig monocytes, it has also been shown that cells
with internalized viral glycoproteins are protected against
antibody-dependent, complement-mediated cell lysis (Van de
Walle et al., 2003). Whether this is also true for an FIPVinfected monocyte will be investigated in the near future.
In conclusion, it can be stated that surface-expressed viral
proteins in FIPV- and FECV-infected monocytes are internalized upon FCoV-specific antibody addition in a very
efficient manner. This internalization does not occur spontaneously; nor can it be induced by non-specific antibodies.
These findings might lead to new insights in strategies for
immune evasion developed by feline coronaviruses.
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Acknowledgements
We are grateful to Dr Hohdatsu and Dr Egberink for supplying
antibodies. H. L. D. and E. C. were supported by the Institute for the
Promotion of Innovation through Science and Technology in Flanders
(IWT-Vlaanderen).
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